南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (4): 498-508.doi: 10.12122/j.issn.1673-4254.2022.04.05

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RUNX3调控胃癌细胞对曲妥珠单抗耐药的机制：基于超效高液相色谱-四极杆/静电场轨道阱质谱的代谢组学分析

刘文虎，汤建才，常晋霞   

  1. 川北医学院药学院，基础医学与法医学院，四川 南充 637100
  • 出版日期:2022-04-20 发布日期:2022-04-29

RUNX3 regulates trastuzumab resistance of gastric cancer cells: a metabolomic analysis based on UPLC-Q Exactive Focus Orbitrap mass spectrometry

LIU Wenhu, TANG Jiancai, CHANG Jinxia   

  1. Department of Pharmacy, School of Basic Medical Sciences & Forensic Medical, North Sichuan Medical College, Nanchong 637100, China
  • Online:2022-04-20 Published:2022-04-29

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摘要： 目的 探讨Runt相关转录因子3（RUNX3）在胃癌曲妥珠耐药细胞中的代谢调控作用，阐释敲除RUNX3逆转耐药的代谢机制。方法 基于超高效液相色谱-四极杆/静电场轨道阱质谱联用技术，对曲妥珠耐药胃癌细胞（NCI N87R）及RUNX3敲除细胞（NCI N87R/RUNX3）进行代谢组分析；采用多变量结合单变量分析方法及二级质谱离子匹配打分筛选差异变量；使用MetaboAnalyst 5.0数据库进行通路富集分析；基于OmicsNet数据库构建差异代谢物-基因调控关系；通过试剂盒检测还原性/氧化性谷胱甘肽（GSH/GSSG）及还原性/氧化性辅酶II（NADPH/NADP）的比值。结果 RUNX3敲除前后NCI N87R细胞的代谢特征显著改变，鉴定到81个对分类有显著贡献的差异代谢物，其中43个代谢物在NCI N87R/RUNX3细胞中增加，38个代谢物减少。RUNX3敲除导致耐药细胞8条通路显著改变（P<0.01），包括谷氨酰胺、糖酵解、甘油磷脂酸-烟酰胺及谷胱甘肽代谢等。试剂盒检测结果显示，NCI N87R/RUNX3胞内GSH/GSSG及NADPH/NADP降低（P<0.01）；差异代谢物-基因网络揭示了代谢分子与基因之间的调控关系。结论 RUNX3通过调控能量代谢及氧化-还原稳态逆转胃癌细胞对曲妥珠单抗耐药，提示RUNX3可能是胃癌曲妥珠单抗耐药的潜在治疗靶标。

关键词: Runt相关转录因子3；曲妥珠抗药性；代谢组学；谷氨酰胺代谢；谷胱甘肽代谢；超高效液相色谱

Abstract: Objective To explore the role of Runt-related transcription factor 3 (RUNX3) in metabolic regulation of trastuzumab-resistant gastric cancer cells and investigate the mechanism of RUNX3 knockdown-mediated reversal of trastuzumab resistance. Methods We performed a metabolomic analysis of trastuzumab-resistant gastric cancer cells (NCI N87R) and RUNX3 knockdown cells (NCI N87R/RUNX3) using ultra performance liquid chromatography (UPLC) coupled with Q Exactive Focus Orbitrap mass spectrometry (MS). Multivariate combined with univariate analyses and MS/MS ion spectrums were used to screen the differential variables. MetaboAnalyst 5.0 database was employed for pathway enrichment analysis. Differential metabolites-genes regulatory relationships were constructed based on OmicsNet database. The changes in GSH/GSSG and NADPH/NADP ratios in NCI N87R/RUNX3 cells were measured using detection kits. Results The metabolic profile of NCI N87R cells was significantly altered after RUNX3 knockdown, with 81 differential metabolites identified to contribute significantly to the classification, among which 43 metabolites were increased and 38 were decreased (P<0.01). In NCI N87R cells, RUNX3 knockdown resulted in noticeable alterations in 8 pathways involving glutamine metabolism, glycolysis, glycerophospholipid, nicotinate-nicotinamide and glutathione metabolism, causing also significant reduction of intracellular GSH/GSSG and NADPH/NADP ratios (P<0.01). The differential metabolites-genes network revealed a regulatory relationship between the metabolic molecules and genes. Conclusion RUNX3 reverses trastuzumab resistance in gastric cancer cells by regulating energy metabolism and oxidation-reduction homeostasis and may serve as a potential therapeutic target for trastuzumab-resistant gastric cancer.

Key words: Runt-related transcription factor 3; trastuzumab resistance; metabolomics; glutamine metabolism; glutathione metabolism; ultra performance liquid chromatography